1. Field of the Invention
The present invention is directed to methods of obtaining lignan from plant materials, particularly flax and flaxseed. The invention is also directed to compositions containing lignan obtained from the methods described herein.
2. Background Art
Lignans are secondary plant metabolites, which are produced from shikimic acid via the phenylpropanoid pathway. They develop from flavonoid precursors and are responsible for conferring resistance to plants against pathogens and predators. Lignans are defined to be compounds possessing a 2,3-dibenzylbutane structure and include matairesinol, secoisolaricinesinol, lariuresinol, isolariciresinol, nordihydroguaiaretic acid, pinoresinol, olivil and other compounds, and modifications thereof, including diglucosides such as but not limited to herbacetin 3,8-0-diglucopyanoside, herbacetin 3,7-0-dimethyl ether and Kaempferol 3,7-0-diglucopyranoside. Diglycerides are known precursors of two important mammalian lignans dibenzylbutyrolactone enterolactone and dibenzyl butane enterodiol (Setchell, et al., Biochem J. 197:447-458 (1981)).
Flaxseed (Linum usitatissimum) is potentially the richest source of phytoestrogens including lignans. The primary lignan found in flaxseed is 2,3-bis (3-methoxy-4-hydroxybenzyl) butane-1,4-diol (secoisolariciresinol) which is stored as the conjugate secoisolariciresinol diglucoside (SDG) in its native state in the plant. Flax seed contains levels of these phytoestrogens which are 75-800 times greater than any other plant food. The plant lignan, catecholic nordihydroguaiaretic acid, is a potent antioxidant previously used by the food industry.
Plant phenolic compounds occur as free monomers or in combination with other phytochemicals, thereby forming esters or glycosides. Phenolic acids are known to have antioxidant activity. The major phenolic constituents of flaxseed are reported to be cournaric acid (4-glucosyl-cinnamic acid) caffeic acid (3-hydroxy-4-glucosyl-cinnamic acid), ferulic acid (3-methoxy-4glucosyl cinnamic acid) and hydroxy methyl glutamic acid. These compounds have antioxidant and hyperchiolesterernic properties.
Numerous reports in literature have documented the phytochemical benefits of flaxseed lignans. Rickard et al. reported that feeding purified lignan at 5% flaxseed diet levels significantly reduces colon and mammary carcinogenesis in animals (Proceedings of the 57the Flax Institute of the United States, (Fargo, N.D.): 8-13 (1998)). Demark-Wahnefried et al. also reported that flaxseed supplementation may have a beneficial effect on prostate cancer biology (Demark-Wahnefried et al., Adult Urology 58(1): 47-52 (2001)).
Additionally, it has been reported that lignans prevent the development of Type I and Type II diabetes by 71% (Prasad, K. Proc. of the American Diabetes Association, (1999)), act as a hypotensive agent with ability to lower blood pressure without affecting heart rate (U.S. patent application Ser. No. 60/140,972, filed Jun. 16, 1999), provide benefits against Lupus Nephritis (U.S. Pat. No. 5,827,256), and reduce development of hypercholesterolemic atherosclerosis in animals (Atherosclerosis 132: 69-76 (1997)), along with numerous reports on the potential antioxidant (Mol. & Cell. Biochem., 202:91-100 (1999)) and anticancer properties (Anticancer Research 18:1405-1408 (1998)).
Flaxseed, in whole, ground or defatted form has been incorporated into animal feeds and food products such as breads, cookies, bagels and muffins. It has also been used for supplementing fiber levels in meat products (WO 00/19842). However, the amounts which can be used are regulated since high oil content of flax and the presence of mucilage contribute to excessive caloric intake and laxation (WO 96/30468).
One of the major problems associated with using flax in foods is the toxicity associated with cyanogenic glycosides present in flaxseed. Cyanogenic glycosides are nitrogenous secondary plant metabolites which if consumed in excess over a long period of time can result in goitrogenic problems and damage to human organs. These glycosides are important natural toxicants in both animal and human nutrition. They have been associated with flaxseed's unique property of protecting animals against the toxic effects of ingested selenium (Smith, et al., J. Org. Chem., 45:507-510 (1980)). The major cyanogenic glycosides present in flaxseed are linustatin, neolinustatin and linamarin with linustatin accounting for 54-76% of the total cyanogenic glycoside content. Defatting of flax meal with hexane is known to produce an enrichment of all individual cyanogenic glycosides on equal weight basis in the meal (Maaza & Oomah, in Flaxseed in Human Nutrition, Cunnane & Thomson eds. AOCS Press, Champaign, Ill. 1995). Therefore, it is important to separate the cyanogenic glycosides from other compounds present in flax.
Methods for the preparation of lignans and other phenolic compound have been reported in literature. In 1956 Bakke and Klosterman described a process for extracting lignan from defatted flax using methanol dioxane (Proceedings of the North Dakota Academy of Sciences 10:18-22 (1956)). However, lignans are known to occur as a “complex” in flax with cinnamic acid glucosides and other compounds. Hence, lignans have originally been referred to as a “polymer” in flax. Sodium and barium methoxides have been used for methanolysis to release lignans free of other compounds (Blake & Klosterman; Thompson et al., Nutr. Cancer 26:59-165 (1996)). Almost all of the lignans present in flaxseed occur as components of a soluble ester-linked complex and do not occur as free glycosides or aglycone. (Muir et al., Proc. of the 58the Flax Institute of the US, Fargo, N.D., 1999). A detailed review of the various methods of extracting lignan and cinnamic acids can be found in Muir, supra. U.S. Pat. No. 5,705,618 and PCT Applications WO 96/30468 and WO 00/78771 also described methods of preparing lignan containing complexes. However, these processes suffer from drawbacks in that they cannot be scaled up for commercialization due to difficulty in achieving the desired separation and purity without involving complex solvent systems, such as ethyl acetate/water, and are difficult to separate using chromatographic techniques or employ expensive methods such as size exclusion chromatography.